CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

450

Using Nanobodies as an Intracellular Biological to Block HIV-1 Through Innate Antiviral Mechanisms Florence M. Stel 1 , Esther M. Zijlstra-Willems 1 , Charlotte E. J. Verkuijlen 1 , Leanne C. Helgers 2 , Killian Vlaming 1 , Neeltje Koostra 1 , Theo B. H. Geijtenbeek 1 1 Amsterdam University Medical Centers, Amsterdam, Netherlands, 2 Academic Medical Center, Amsterdam, Netherlands Background: HIV-1 has been a global health problem for decades, and finding a cure remains a challenge. Recent studies have explored antibodies targeting various parts of HIV as alternative therapies. These antibodies bind the virus and enter the cell, where the complex is sensed and targeted for proteasomal degradation by the E3 ubiquitin ligase and antibody receptor TRIM21. Here we investigated the potential of this TRIM21 technology to develop intracellular immunity against HIV-1 infection. Our hypothesis is that intracellular recognition of HIV-1 by antibodies results in degradation of viral particles. Methods: We designed nanobodies, based on llama antibodies, targeting the HIV-1 capsid protein p24, fused to the human IgG1 Fc domain. These p24 nanobody-Fc constructs were synthesized and produced by a mammalian expression system. Next, they were characterized by flow cytometry and tested for their functionality to bind p24 through an ELISA-based binding assay. Lastly, p24 nanobody-Fc constructs were stably expressed intracellularly in the U87-CD4-CCR5 cell-line. Engineered cells were infected with the HIV-1 strain NL4.3-BaL. Moreover, Fc-modified nanobodies were purified using column chromatography and transfected into cells previously infected with NL4.3-BaL. Quantification of p24 was done using ELISA and flow cytometry. Statistics were performed using two-way ANOVA. Results: Our data show that our Fc-modified nanobodies recognize intact HIV-1 capsid and have a functional Fc domain. Notably, HIV-1 p24 capsid was neither detected intracellularly nor extracellularly after HIV-1 exposure in the p24 nanobody-Fc expressing cell-line, even though HIV-1 had integrated in the genome at levels comparable to control cells. These results demonstrate that the nanobodies are blocking production of new virus particles, rather than targeting HIV-1 during initial infection. In cells transfected with the purified nanobody a similar decrease of p24 was observed. These data strongly suggest that the Fc-modified p24 nanobody actively degrades p24 and thereby prevents new virus production. Conclusions: To conclude, we have developed a novel p24 nanobody construct that efficiently prevents HIV-1 production. Intracellular production, as well as transfection of Fc-modified p24 nanobodies highly reduces p24 production levels in U87-CD4-CCR5 cells upon infection with HIV-1. This technology holds great promise for the engineering of therapeutic antibodies to prevent viral infections via targeting of host or viral proteins. Viral Genetic Traits of Durable Control in Dual Immunotherapy-Treated SHIV-Infected Rhesus Macaques Manukumar Honnayakanahalli Marichannegowda 1 , So-Yon Lim 2 , Jina Lee 2 , Brandon Keele 3 , James Whitney 2 , Satish Pillai 1 1 Vitalant Research Institute, San Francisco, CA, USA, 2 Boston College, Chestnut Hill, MA, USA, 3 Frederick National Laboratory for Cancer Research, Frederick, MD, USA Background: A recent study demonstrated that combined administration of N-803 (IL15 agonist) and the 10-1074 and 3BNC117 broadly neutralizing antibodies (bNAbs) results in durable virologic remission following ART cessation in SHIV-infected, ART-suppressed rhesus macaques (RM). Here, we investigate the impact of this curative intervention on viral population genetics to identify SHIV genetic features associated with durable remission. Methods: Plasma samples were collected during analytical treatment interruption (ATI) from SHIV-AD8-infected ART-suppressed RM treated with N-803 and the 10-1074 and 3BNC117 bNAbs (N=8) and SHIV-AD8-infected ART suppressed RM control animals (N=8). Viral RNA was extracted, and multiple SHIV env gene sequences were obtained using single genome amplification (SGA). Phylogenetic reconstruction, pairwise distance calculations, entropy calculations, and highlighter plots were used to compare viral genetics and protein features between groups. Signatures of natural selection were analyzed using SNAP and HyPhy software (maximum likelihood method). Results: A total of 306 SHIV env gene sequences (152 from controls and 156 from treated animals) were generated (Figure 1). Viral genetic diversity was higher in immunotherapy-treated animals with respect to controls (p<0.0001, t test). Genetic divergence from the SHIV-AD8 ancestral inoculum sequence The figure, table, or graphic for this abstract has been removed.

neutralizers in late infection. This indicates that although NK cell cytotoxicity may be associated with favorable immune responses in acute HIV infection, when antibody responses are forming and breadth is developing NK cell activity may be detrimental. This was validated by cyTOF data where, in early infection, broad neutralizers expressed higher levels of HLA-DR at baseline and IFNγ upon stimulation indicating early activation. In late infection NK cells from narrow neutralizers expressed higher levels of CCR7, CXCR3, CXCR5, CD107a and TRAIL, indicating NK trafficking to lymphoid tissue where they could limit antibody responses as well as cytotoxic potential. Conclusions: Here we find that NK cells have differential phenotypes in broad and narrow neutralizers. In early infection NK cells from broad neutralizers are more activated, but by late infection timepoints, NK cells from narrow neutralizers show greater activation. This flip-flop in NK cell function during the course of HIV infection could indicate that NK cell responses may be favorable in the context of early untreated infection for their natural antiviral activity. However, as infection progresses, NK cell activity may be detrimental when it comes to antibody development, which could similarly affect vaccine responses. Combination of eCD4-IgG1 Delivered by AAV9 and AZD5582 in SIV-Infected, ART-Suppressed Infant RM Jairo A. Fonseca 1 , Alexis C. King 1 , Omotayo Farinre 1 , Shan Liang 2 , Lucas Alves Britto Da Costa 3 , Adrian Khoei 4 , Stephanie Ehnert 2 , Jennifer S. Wood 2 , Matthew R. Gardner 1 , Koen K. Van Rompay 5 , Jeffrey D. Lifson 6 , Mackenzie Cottrell 4 , Mauricio A. Martins 7 , Ann M. Chahroudi 1 1 Emory University, Atlanta, GA, USA, 2 Emory National Primate Research Center, Atlanta, GA, USA, 3 University of Florida, Gainesville, FL, USA, 4 University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, 5 University of California Davis, Davis, CA, USA, 6 Frederick National Laboratory for Cancer Research, Frederick, MD, USA, 7 The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, Jupiter, FL, USA Background: Latency reversal and immune-mediated clearance is a cure strategy based on eliminating HIV-infected CD4+ T cells during ART suppression. Here, we used AZD5582 to stimulate the non-canonical NF-κB pathway to reverse latency, as previously shown in adult rhesus macaques (RM). To target the reactivated reservoir via antibody-mediated cellular cytotoxicity, we used AAV9-delivered eCD4-Ig, a fusion protein comprising the CD4 ectodomain, an IgG1-Fc, and the TYR-sulfated regions of CCR5 that bind to HIV/ SIV Env on the cell surface. Methods: 20 infant RM (10 male, 10 female) with no/low levels of AAV9 neutralizing antibodies were selected after screening. At 4 weeks old, the animals were orally infected with SIVmac251 and started on ART 21 days post-infection. Half received an IM injection of AAV9-eCD4-IgG1 on the day of ART initiation. AZD5582 was administered at 40 weeks post-infection. The dose was based on a pharmacokinetic model as prior work showed reduced latency reversal in infants vs adults. On-ART viremia and viral rebound dynamics following ART interruption were monitored. Results: eCD4-IgG1 expression was >10 µg/mL in 10/10 RM, with sustained levels for at least 59 weeks. One animal developed transient anti-drug antibodies, which did not affect long-term eCD4-IgG1 expression. During AZD5582 treatment, 8/10 infant RM had on-ART viremia >60 copies/mL, peaking at 5600. Of 67 viral load measurements taken, 47 (65.7%) were above baseline, with significantly higher on-ART viremia in the intervention group versus controls (p=0.0147). 6 RM from each group underwent ART interruption. Despite continued eCD4-IgG1 expression, all treatment RM rebounded between 6-11 days off ART. 2/6 RM in the intervention group experienced a ~4.5 log decline in viremia post-rebound, which was not observed in controls. Partial correlation analyses indicated a negative correlation between peak AZD5582 induced on-ART viremia and the AUC of the viremia during ART interruption (r = -0.870, p = 0.055) Conclusions: This study is an important proof-of-concept, as it is the first to combine an AAV9-delivered biologic with an LRA in infants. AAV9's long-lasting transgene expression makes it a promising delivery platform for HIV neutralizing molecules, including bNAbs. Peak levels of on-ART viremia during AZD5583 treatment surpassed those observed in prior studies of adult RM. Further research is needed to elucidate the mechanisms behind post-rebound viral control in the intervention group.

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Poster Abstracts

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CROI 2025 110